Pulmonary surfactant is a lipid-protein complex which reduces surface tension at the air-liquid interface in the lung, preventing alveolar collapse and allowing breathing. Deficiency in pulmonary surfactant causes respiratory distress syndrome (RDS), a major cause of neonatal morbidity and mortality. Surfactant preparations routinely used for treatment of RDS contain phospholipids and surfactant proteins SP-B and SP-C. Homeostasis of pulmonary surfactant involves rapid uptake and recycling of surfactant by pulmonary epithelial cells. The long term aim of this research is to delineate the signals and processes governing the homeostasis of pulmonary surfactant. The present proposal will test the hypothesis that the stoichiometry of surfactant proteins and palmitoylation of SP-C within distinct subfractions of surfactant determine their size and structure, in turn determining the mechanism of their uptake by pulmonary type II epithelial cells, and their intracellular routing along recycling or degradative pathways. The uptake and intracellular routing of labeled SP-C will be assessed in type II cells and in a cell line derived from type II cells (MLE-12 cells). The role of palmitoylation of SP-C, SP-A and SP-B in uptake and intracellular routing of SP-C will be determined. Preliminary experiments demonstrated that lipid uptake is stimulated by SP-C and proceeds by mechanisms involving both endocytosis of intact vesicles and lipid mixing with the plasma membrane. The influence of the stoichiometry of SP-A, SP-B, and SP-C on these mechanisms of uptake and the recycling pathway of lipids in MLE-12 cells will be determined using fluorescent lipid probes. The uptake and intracellular fate of SP-G and lipids associated with heavy and light surfactant subfractions will be correlated with the stoichiometry of the surfactant proteins within discrete subfractions. Molecular interactions among surfactant proteins and lipids form the basis for tubular myelin structure and biophysical activity. The degree of oligomerization of SP-B in surfactant, and whether it interacts directly with SP-C will be assessed using fluorescence energy transfer between labeled SP-B and SP-C in lipid vesicles. The specific sites of interaction between SP-A and SP-B will be assessed using phototransfer labeling of SP-A by derivatized SP-B in reconstituted tubular myelin. Knowledge of the signals governing uptake, recycling and catabolism of pulmonary surfactant is essential to the treatment of lung diseases including RDS and alveolar proteinosis.